Fluorometry could protect valuable vineyards

A method that accurately detects the presence of natural antimicrobial agents in grapevines through fluorescence could aid grape growers in protecting their valuable crops from a variety of potentially destructive pathogens.

Although techniques such as high-performance liquid chromatography already are used for this purpose, the approach is time-consuming, destructive to the plant and lacking information about spatial distribution.

The new method, tested by researchers in France, has the potential to provide earlier and quicker detection. Using commercially available spectrofluorometers, they assessed stilbene synthesis in grapevine leaves on various scales of the leaf simultaneously. Stilbenes, small polyphenolic molecules, are considered a grapevine’s most important defense against pathogens such as downy mildew. Naturally induced by a host of factors, including climate, fungus infections and exposure to UV light, their presence is an early indication of potential problems. Upon exposure to UV light, stilbenes emit an intense blue fluorescence.

The study was conducted jointly by the Institut National de la Recherche Agronomique in Colmar and by the Centre National de la Recherche Scientifique in Orsay. The team, led by Anne Poutaraud, used greenhouse-grown Muscat Ottonel leaves for its research. The samples were subjected to four different treatments to produce various stilbene levels: a control that received no treatment, inoculation of the leaves with the pathogen P. viticola, exposure to UVC light for seven minutes and exposure to the same light for 14 minutes.

The scientists used a Varian spectrofluorometer on each set of leaves to measure excitation and emission fluorescence spectra to find the best wavelengths and a Tecan microplate fluorometer to read how the fluorescence was distributed on the leaf. A Zeiss inverted epifluorescence microscope outfitted with a Zeiss CCD camera took images of the samples.

Researchers developed a fluorometry technique that can be used to detect the early presence of potentially destructive pathogens in grapevines.Results showed the highest fluorescence (and therefore the highest active stilbene content) on the two sets of leaves treated with UV light. Both sets of leaves, which had a peak emission of 390 nm, achieved fluorescence levels of more than 1000 quinine sulfate equivalent units on their abaxial side. The inoculated leaves, by comparison, achieved more than 400 quinine sulfate equivalent units. The control leaves exhibited no fluorescence.

To gauge the effectiveness of the fluorometry, the researchers tested the same leaves using high-performance liquid chromatography. The results from the two procedures were similar: Readings depended on the type of exposure the leaf was subjected to with chromatography, mirroring the findings from the fluorescence experiment. High stilbene content was measured in the leaves exposed to UV light, whereas moderate amounts were found in the inoculated leaf. No traces were found in the control.

The researchers said the results show that measurement of UV-induced blue fluorescence correlates with the stilbene content measured by chromatography. The fluorescence method is suitable for prescreening crops, they said, although high-performance liquid chromatography would still be needed for a more detailed analysis once a problem is detected.

The team plans further testing to measure whole leaves in vivo in an outdoor agricultural setting to see whether the same behavior is observed.

The emission of light or other electromagnetic radiation of longer wavelengths by a substance as a result of the absorption of some other radiation of shorter wavelengths, provided the emission continues only as long as the stimulus producing it is maintained. In other words, fluorescence is the luminescence that persists for less than about 10-8 s after excitation.